Capillary electrophoresis (CE) systems use electric fields to separate molecules within narrow-bored capillaries while the capillaries are filled with conductive buffers or gel matrix. Samples are injected into the capillary tubing for separation under the high electric field or hydrodynamic flow. Sample molecules are detected by different means while passed through the detection window. UV absorption detection is the most common detection means since almost all analytes have UV absorption property. However, because of the use of narrow-bored capillaries (≦100 μm I.D.) for absorption measurement and the measurement of a small light intensity change from a high background light source, sensitivity is limited at 10−5 M levels. Laser induced fluorescence (LIF) detection can also be used in capillary electrophoresis for samples that naturally fluoresce or are chemically modified to contain fluorophores. LIF provides much higher sensitivity than the UV absorption detection due to the ultra low background. However, the use of laser as excitation light source in fluorescent detection is expensive and difficult to maintain. Alternatively, Xenon lamp or light emitted diode (LED) have been used for fluorescent excitation light source.
In order to improve the sample throughput, multiple capillaries are used to analyze multiple samples simultaneously, these multiplexed capillary array electrophoresis systems are used in many commercial DNA sequencers. Most of them use a laser as the light source, including confocal scanning laser induced fluorescence (e.g. U.S. Pat. No. 6,270,644), sheath flow detectors (e.g. U.S. Pat. Nos. 5,468,364 and 6,048,444), side-entry optical excitation geometry (e.g., U.S. Pat. Nos. 5,582,705 and 5,741,411), and fiber optics for excitation and emission collection (U.S. Pat. No. 6,870,165).
There is a need for multiple wavelength measurement during fluorescent detection in CE. For example, DNA sequencing determination in the commercial DNA sequencers requires the measurement of four different wavelength regions to discriminate four different nucleotide bases for sequence determination since each type of nucleotide is labeled with a different fluorescent tag. The dominant signal from different wavelength regions determines the nucleotide base. In some cases, a filter wheel has been used to measure different wavelength regions sequentially by rotating the filter wheel to the desired filters. This method is not efficient since only one wavelength region can be measure at any given time. U.S. Pat. Nos. 6,461,492 and 6,554,986 uses beam splitters to divide the light emission into multiple beams with multiple detectors for multi-wavelength detection. This method measures only one capillary signal at a time and requires a scan of each detection window sequentially for multiplexed capillary array electrophoresis operation. In addition, this method requires the use of multiple detectors for measurement which increases the cost and maintenance substantially. U.S. Pat. No. 6,048,444 reveals a method that used a single detector to measure four different wavelength regions simultaneously. The fluorescent signal is split by an image splitter prism and projected into a two-dimensional detector. Wavelength is selected by using four different filters. However, since the fluorescent emission is split to four regions before filtering, it suffers the low light detection efficiency. U.S. Pat. No. 5,998,796 discloses a method of using a transmission grating for multi-wavelength analysis for multiplexed capillary array electrophoresis. U.S. Pat. No. 5,741,411 revealed a method using a tilted filter to split the fluorescent signal into two for two wavelength fluorescent measurements while using a single two-dimensional detector. However, this method was limited to two wavelength regions measurement.
As can be seen from the state of the art, it would be desirable to develop a low-cost, less complex design resulting in high light detection efficiency, high throughput in multiple-capillaries, and multi-wavelength detection in multiplexed capillary array electrophoresis systems for biological separation to overcome the limitations in the prior art.